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Journal of the Formosan Medical Association (2013) xx, 1e6
Available online at www.sciencedirect.com
journal homepage: www.jfma-online.com
ORIGINAL ARTICLE
Chemokine MCP1/CCL2 and RANTES/CCL5 gene polymorphisms influence ¨nlein purpura susceptibility HenocheScho and severity Hsin-Hui Yu a, Pi-Hua Liu b, Yao-Hsu Yang a, Jyh-Hong Lee a, Li-Chieh Wang a, Wei-J. Chen c,d, Bor-Luen Chiang a,e,* a
Department of Pediatrics, National Taiwan University Hospital, Taipei, Taiwan, ROC Clinical Informatics and Medical Statistics Research Center, Chang Gung University, Tao-Yuan, Taiwan, ROC c Institute of Epidemiology, College of Public Health, National Taiwan University, Taipei, Taiwan, ROC d Genetic Epidemiology Core Laboratory, Research Center for Medical Excellence, National Taiwan University, Taipei, Taiwan, ROC e Department of Medical Research, National Taiwan University Hospital, Taipei, Taiwan, ROC b
Received 21 June 2012; received in revised form 26 November 2012; accepted 21 December 2012
KEYWORDS chemokine; HenocheScho ¨nlein Purpura (HSP); MCP1/CCL2; polymorphism; RANTES/CCL5
Background/Purpose: HenocheScho ¨nlein purpura (HSP) is the most common small vessel vasculitis in children. It is considered to be an IgA-containing immune complex-mediated disease. Chemokines are small secreted proteins that attract leukocytes during inflammation. Our aim was to determine the serum levels of chemokines and investigate the association of chemokine gene polymorphisms with childhood HSP. Materials and methods: Serum levels of chemokines (interleukin-8/CXCL8, MCP-1/CCL2, RANTES/CCL5, MIG/CXCL9, and IP-10/CXCL10) were determined using cytometric beads arrays. We investigated the association of three single-nucleotide polymorphisms (SNPs) MCP1/CCL2 2518C/T, RANTES/CCL5 403C/T, and RANTES/CCL5 28C/G with HSP in 85 HSP patients and 136 healthy controls. Results: Five serum chemokine levels were significantly elevated in patients with the acute stage of HSP compared to the normal controls (p < 0.05). MCP1/CCL2 2518 TT genotype and T allele were associated with the risk for HSP with OR (95% CI) 3.32 (1.45e7.59) and 1.78 (1.20e2.64), respectively. The RANTES/CCL5 28 GG genotype was associated with a significantly lower percentage of corticosteroid usage and lower corticosteroid accumulative
* Corresponding author. Department of Medical Research, National Taiwan University Hospital, Number 7, Chung-Shan South Road, Zhongzheng District, Taipei City 100, Taiwan, ROC. E-mail address: [email protected] (B.-L. Chiang). 0929-6646/$ - see front matter Copyright ª 2013, Elsevier Taiwan LLC & Formosan Medical Association. All rights reserved. http://dx.doi.org/10.1016/j.jfma.2012.12.007
Please cite this article in press as: Yu H-H, et al., Chemokine MCP1/CCL2 and RANTES/CCL5 gene polymorphisms influence HenocheScho ¨nlein purpura susceptibility and severity, Journal of the Formosan Medical Association (2013), http://dx.doi.org/10.1016/ j.jfma.2012.12.007
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H.-H. Yu et al. dose in HSP patients. RANTES/CCL5 403 TC and TT genotype were significantly associated with renal manifestations with an OR (95% CI) of 4.33 (1.44e12.99), adjusted for sex and age and the other two SNP genotypes. Conclusion: Our results support the fact that chemokines play important roles in the pathogenesis of HSP. MCP1/CCL2 gene polymorphisms were associated with susceptibility for HSP. RANTES/CCL5 gene polymorphisms may be related to disease severity and HSP nephritis. Copyright ª 2013, Elsevier Taiwan LLC & Formosan Medical Association. All rights reserved.
Introduction HenocheScho ¨nlein purpura (HSP) is the most common small vessel vasculitis that occurs in children. It is an IgAcontaining immune complex-mediated vasculitis, which is triggered by common viral or bacterial infections.1 The European League Against Rheumatism (EULAR) proposed consensus criteria for the classification of HSP in 2010, which require individuals to have purpura or petechiae with lower limb predominance and at least one of the four following criteria: abdominal pain, histopathology (typical leukocytoclastic vasculitis or proliferative glomerulonephritis with predominant IgA deposits), arthritis or arthralgia, or renal involvement (proteinuria > 0.3 g/day or hematuria or red blood cell casts >5 red blood cells/high power field, or 2þ on a dipstick).2 Perivascular accumulation of neutrophils in the acute inflammatory lesions is the typical histopathological feature of HSP, known as leukocytoclastic vasculitis. Lymphocyte and monocyte/macrophage infiltration and neovascularization are found later in the disease course.3 The mechanisms underlying the perivascular leukocyte infiltrates and endothelial damage in HSP are not fully understood. Previous studies have shown the elevation of inflammatory mediators in the serum of HSP patients, including tumor necrosis factor (TNF)-a, interleukin-6, transforming growth factor (TGF)-b, and vascular endothelial growth factor (VEGF).47 Genetic variations in key inflammatory genes, including interleukin-1 beta (IL1B), interleukin-1 receptor antagonist (IL1RA), intercellular adhesion molecule-1 (ICAM1), IL8, VEGF, and TGFB gene polymorphisms also contribute to the susceptibility or different clinical manifestations in HSP.813 Proinflammatory chemokines are a group of small secreted proteins that attract leukocytes to the inflammatory site and then activate them. It is a critical pathogenic factor in tissue damage during the development of vasculitis. Chemokine monocyte chemoattractant protein 1 (MCP-1/CCL2), and interferon gamma-induced protein-10 (IP-10/CXCL10) are strong chemoattractants for monocytes/macrophages. Regulated upon activation, normal Tcells expressed and secreted RANTES/CCL5, monokine induced by gamma interferon (MIG/CXCL9), and IP-10/ CXCL10 are chemoattractants for T cells.14 The roles of chemokines have been investigated in many rheumatic diseases and vasculitis, such as Kawasaki disease, Behcet’s disease, and polymyalgia rheumatica.1517 However, the roles of proinflammatory chemokines except IL-8 have not been investigated in HSP before. We therefore aimed to investigate the changes in serum levels of five different proinflammatory chemokines during
the acute and convalescent stages of HSP. We tested the association between serum chemokines levels at the acute stage of HSP and disease severity. We also tested the hypothesis that single nucleotide polymorphisms (SNPs) in the MCP1/CCL2 and RANTES/CCL5 genes would be associated with serum chemokine levels, disease susceptibility, or severity.
Materials and methods Study subjects This study was conducted at National Taiwan University Hospital, a tertiary referral center. Eighty-five patients who were diagnosed with HSP, and 136 volunteersdhealthy, sex-matched children (73 boys and 63 girls) aged 2e18 yearsdwere enrolled. The diagnosis of HSP was based on the criteria defined by EULAR in 2010. Disease severity was clinically assessed by the degree of systemic involvement. Gastrointestinal involvement was defined as abdominal pain with/without vomiting, and/or stool occult blood 1þ, and/or melena. Renal involvement was defined as per the EULAR criteria of 2010. Informed consent and institutional approval were obtained.
Serum and cytometric bead array Serum was collected from 63 of the 136 age-matched healthy controls, and 55 of 85 HSP patients in the acute stage of HSP. Twenty HSP patients also donated blood during the convalescent stage of HSP. The convalescent stage was defined as the stage in which there was complete resolution of symptoms and signs for 1 month after discontinuing medication. Serum concentrations of MIG/ CXCL9, IP-10/CXCL10, MCP-1/CCL2, RANTES/CCL5 and IL8/CXCL8 were analyzed by Human Chemokine Kit I Cytometric Beads Array kits from BD Biosciences (San Diego, CA, USA; BD CBA), according to the manufacturer’s recommended protocols. The serum samples for RANTES/CCL5 were diluted 1:100 using the assay diluent to fit the concentration range.
DNA extraction and single nucleotide polymorphism (SNP) genotyping Genomic DNA of the 85 HSP patients and 136 controls was extracted from the anticoagulated blood using a commercially available DNA extraction kit (QIAGEN, QIAamp DNA Blood Mini Kit, Germany) and stored at 20 C
Please cite this article in press as: Yu H-H, et al., Chemokine MCP1/CCL2 and RANTES/CCL5 gene polymorphisms influence HenocheScho ¨nlein purpura susceptibility and severity, Journal of the Formosan Medical Association (2013), http://dx.doi.org/10.1016/ j.jfma.2012.12.007
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Chemokine MCP1 and RANTES gene polymorphisms in HSP before testing. We used the candidate gene approach by selecting well-investigated SNPs in chemokine genes (MCP1/CCL2 2518 C/T (dbSNP ID rs1024611), RANTES/ CCL5 403 C/T (dbSNP ID rs2107538), and RANTES/ CCL5 28 C/G (dbSNP ID rs2280788) in a case control study design. We chose these three previously identified common functional variants from the National Center for Biotechnology Information (NCBI) database (http://www.ncbi. nlm.nih.gov/gene). Genotypes were identified by multiplex polymerase chain reaction (PCR) and SNP analyses with the GenomeLab SNPstream genotyping platform (Beckman Coulter Inc. Fullerton, CA) and its accompanying SNPstream software suite. The primers for the multiplex PCR and single-base extension primers (tagged SNP primers) were designed by the software provided by Beckman Coulter Inc. The detailed methods, reagents, and PCR conditions were described in a previous report.18 The direct sequencing reactions using an ABI 3730 automated DNA sequencer (Applied Biosystems, Foster City, CA) in 10 cases and 10 controls resulted in identical genotypes by multiplex PCR and SNP analyses. The average genotype call rates by multiplex PCR were 95.29% and 98.92%, respectively, for the cases and controls. The overall genotype call rate was 98.32%.
Statistical methods The ManneWhitney U test was used to compare the serum chemokine levels between the groups of patients. A paired t test was used to compare serum chemokine levels in the acute and convalescent stage of HSP. A c2 goodnessof-fit test was performed using the SAS/Genetics module, version 9.1 (SAS Institute, Cary, NC) to assess whether the observed genotype frequencies of each SNP marker departed from the HardyeWeinberg equilibrium (HWE) in the HSP patients and control subjects separately. After the frequencies of the alleles and genotypes had been calculated, Armitage’s trend and genotypic case-control tests were determined with c2 tests using the SAS/Genetics module to assess each SNP for a global association with HSP. When the global association test was significant, an unconditional logistic regression was undertaken using SAS Proc LOGISTIC to calculate allele or genotype odds ratios (OR) with 95% confidence intervals (CIs) for the potential association between HSP and SNPs. Logistic regression models were used to test the association of genotypes with clinical outcomes. A p value of less than 0.05 was considered to be statistically significant. Power calculations were performed at a type 1 error rate (a value) of 0.05 and a prevalence of 0.0001 for HSP using the Genetic Power Calculator (http://pngu.mgh.harvard.edu/ wpurcell/gpc/). The prevalence was estimated from the annual incidence of 12.9 per 100,000 children in Taiwan.19
Results Patient characteristics The demographic data of the 85 children with HSP are summarized in Table 1. All 85 (100%) patients had purpura,
3 Table 1 Clinical characteristics and basic demographic data of the 85 HSP patients. HSP patients Sex (male:female) Age (years) Previous infection history Purpura Gastrointestinal involvement Arthritis or arthralgia Renal involvement Central nervous system involvement Recurrence Steroid treatment Disease duration of 6 months or more IgA elevation
46:39 (1.18:1) 6.4 2.7 51/84 (60.7%) 85/85 (100%) 62/85 (72.9%) 60/85 (70.6%) 30/85 (35.3%) 2/85 (2.4%) 9/85 (10.6%) 77/84 (91.7%) 14/85 (16.5%) 42/70 (60%)
of whom 29 (34.1%) had prolonged purpura for 4 weeks or more. Sixty-two (72.9%) patients had gastrointestinal (GI) involvement, of whom 18 (21.2%) had GI bleeding. Thirty (35.3%) patients had renal involvement, including 14 (16.5%) with severe nephropathy (nephrotic syndrome or impaired renal function) and three (3.5%) with renal sequelae (persistent urine abnormalities over 2 years). Sixty percent of the HSP patients had serum IgA elevation at disease onset.
Chemokine expressions in the HSP patients and controls The serum chemokine levels of MIG/CXCL9, IP-10/CXCL10, MCP-1/CCL2, RANTES/CCL5 and IL-8/CXCL8 were significantly elevated in patients with the acute stage of HSP compared to the normal controls (p < 0.05) (Fig. 1). Compared with the acute stage, serum chemokine levels decreased significantly in the convalescent stage of HSP in 20 patients (p < 0.05), except for IL-8/CXCL8 (p Z 0.109) and MIG/CXCL9 (p Z 0.805) (Fig. 2). There was more renal involvement in the HSP patients with persistently elevated
Figure 1 Chemokine serum concentrations in the controls and HSP patients in the acute stage. The serum chemokine levels of MIG/CXCL9, IP-10/CXCL10, MCP-1/CCL2, RANTES/ CCL5, and IL-8/CXCL8 are significantly elevated in the acute stage of HSP compared to the normal controls (p < 0.05). HSP Z HenocheScho ¨nlein purpura (*p < 0.01, **p < 0.0001).
Please cite this article in press as: Yu H-H, et al., Chemokine MCP1/CCL2 and RANTES/CCL5 gene polymorphisms influence HenocheScho ¨nlein purpura susceptibility and severity, Journal of the Formosan Medical Association (2013), http://dx.doi.org/10.1016/ j.jfma.2012.12.007
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4 MIG/CXCL9 levels in the convalescent stage than that of the HSP patients with decreased MIG/CXCL9 levels in the convalescent stage (71.4% vs. 16.7%) (p Z 0.045).
Polymorphisms and HSP risk Three SNPs and RANTES/CCL5 and MCP1/CCL2 genes were investigated. None of the SNP distributions showed a deviation from the HardyeWeinberg equilibrium. Table 2 presents the genotypes of the three SNPs for HSP patients (n Z 85) and controls (n Z 136), as well as ORs for each of the SNPs. MCP1 2518 TT and the TC genotype carried an increased risk for HSP (OR (95% CI) 3.32 (1.45e7.59) and 2.0 (1.03e3.9), respectively). After adjusting for age and sex, the adjusted OR (95% CI) of MCP1 2518 TT and TC genotype remained unchanged: 3.32 (1.45e7.59) and 2.01 (1.03e3.92), respectively. The MCP1 2518 T allele carried an increased risk for HSP with an allelic OR (95% CI) of 1.78 (1.20e2.64), p < 0.0001.
Chemokine expressions in the HSP patients with different genotypes Serum RANTES/CCL5 and MCP-1/CCL2 levels in the acute stage of HSP did not significantly correlate with the corresponding genotype (data not shown).
Factors associated with clinical outcomes in the HSP patients Patients with the RANTES/CCL5 28 G/G and C/G genotype had a significantly lower percentage of corticosteroid usage and lower corticosteroid accumulative dose (median dose 11.3 mg/kg vs. 20.5 mg/kg) (p Z 0.04) than patients carrying the RANTES/CCL5 28C allele. The RANTES/ CCL5 403 TC and TT genotypes were associated with GI
H.-H. Yu et al. and renal involvement in the HSP patients (OR (95% CI) 2.97 (1.06e8.33) and 2.7 (1.06e6.85), respectively). However, using multivariate logistic regression models adjusting for age, sex, RANTES/CCL5 28G, RANTES/CCL5 403T, and MCP1/CCL2 2518T genotypes, only the RANTES/ CCL5 403 TC and TT genotypes were associated with renal involvement (OR (95% CI) 4.33 (1.44e12.99), p Z 0.009). There was no significant association between clinical outcomes and the RANTES/CCL5 28 GG and CG genotypes (Table 3).
Discussion Our results showed significant elevations of the chemokines MIG/CXCL9, IP-10/CXCL10, MCP-1/CCL2, RANTES/CCL5, and IL-8/CXCL8 in the sera of patients with the acute stage of HSP. Our finding suggests that proinflammatory chemokines, which function in the recruitment of different types of leukocytes, are involved in the pathogenesis of HSP. Our results are consistent with a recent study that showed elevation of serum levels of RANTES/CCL5, CXCL16, CX3CL1 in the acute stage of HSP,20 but contradictory to previous reports of decreased IP-10/CXCL10 serum level or no elevation of IP-10/CXCL10, MCP-1/CCL2, and Gro-a/CXCL1 levels in acute HSP.21,22 Recent studies have shown that chemokines are produced during inflammatory stimuli from endothelial cells. Yang et al found that IgA antiendothelial cell antibodies in the serum of HSP patients could stimulate human umbilical vein endothelial cells (HUVECs) through extracellular signalregulated kinase (ERK) pathways to produce IL-8/CXCL8.6,23 Chen et al further showed that sera from patients with active HSP could induce MCP-1/CCL2, RANTES/CCL5, IL-8/ CXCL8, CXCL16, and CX3CL1 production in HUVECs or the human dermal microvascular endothelial cell line (HMEC-1)
Figure 2 Chemokine serum concentrations in the HSP patients in the acute and convalescent stage. The serum MCP-1, RANTES, and IP-10 levels decrease significantly in the convalescent stage (p < 0.05). HSP Z HenocheScho ¨nlein purpura. Please cite this article in press as: Yu H-H, et al., Chemokine MCP1/CCL2 and RANTES/CCL5 gene polymorphisms influence HenocheScho ¨nlein purpura susceptibility and severity, Journal of the Formosan Medical Association (2013), http://dx.doi.org/10.1016/ j.jfma.2012.12.007
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Chemokine MCP1 and RANTES gene polymorphisms in HSP Table 2
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Association analysis between the SNPs of the MCP1/CCL2 and RANTES/CCL5 genes and HSP.
Gene
SNP genotype
MCP1
2518 C/T
RANTES
403 C/T
RANTES
28 C/G
TT TC CC TT TC CC GG GC CC
HSPa (n Z 85)
Controla (n Z 136)
ORsb
95% CIb
pb
21 44 17 10 36 39 2 15 68
19 66 51 13 54 69 7 22 107
3.32 2.0 1 0.92 1.09 1 0.33 1.15 1
1.45e7.59 1.03e3.90 d 0.34e2.53 0.56e2.12 d 0.03e3.29 0.51e2.62 d
0.005 0.04 d 0.88 0.80 d 0.35 0.73 d
(25.6%) (53.7%) (20.7%) (11.8%) (42.4%) (45.9%) (2.4%) (17.6%) (80%)
(14.0%) (48.5%) (37.5%) (9.6%) (39.7%) (50.7%) (5.1%) (16.2%) (78.7%)
CI Z confidence interval; HSP Z HenocheScho ¨nlein purpura; OR Z odds ratio; SNP: single nucleotide polymorphism. a The number of the patients along with the percentage in brackets. b Logistic regression models are used to calculate OR and 95%CIs, and the corresponding p values for each genotype.
through ERK and nuclear factor-kB (NF-kB) pathways. Patients’ sera-stimulated HMEC-1 supernatants were also noted to enhance leukocytes migration.20 These results support the important role of chemokines in perivascular leukocyte infiltrates and the vascular damage in HSP. The genetic basis of HSP is unclear. To the best of our knowledge, this is the first study to report an association of MCP1/CCL2 2518T and the risk of susceptibility to HSP. MCP-1 expression is inducible by proinflammatory cytokines (TNF-a, IL-1b) through NF-kB in various cell types. The gene expression of the MCP1/CCL2 2518C/T polymorphism in the promoter region is controversial. Kim et al reported that the T allele at 2518 was associated with upregulation of MCP-1/CCL2.24 In contrast, Rovin et al and Gonzalez et al reported that MCP1/CCL2 2518C produced more MCP1.25,26 These discrepancies may be related to differences in disease or ethnicity. The MCP1/CCL2 2518TT genotype has been shown to be an independent risk factor for the progression of renal diseases in Japanese patients with IgA nephropathy, which shares a common disease mechanism with HSP nephritis.27 To elucidate the role of the MCP1/ CCL2 2518 gene polymorphism in HSP, the relationship between the MCP1/CCL2 2518 genotype, MCP-1 production from endothelial cells, and macrophage infiltration in skin and other target organs such as the kidney should be investigated in future studies. Our results showed that the RANTES/CCL5 403 TC and TT genotypes carried an increased risk of HSP nephritis. RANTES/CCL5 403 C to T change results in a new binding element for GATA transcriptional factors and has higher transcriptional activity.28 The promoter of that RANTES/
Table 3
Frequency and ORs of RANTES/CCL5 genotypes in HSP patients with different disease manifestations.
HSP involvement
SNP
GI
RANTES RANTES RANTES RANTES
Renal
CCL5 28 gene polymorphism site is close to the NF-kB binding site, which is located at position 32 and plays a critical role in the upregulation of RANTES promoter activity. Liu et al reported that RANTES/CCL5 28G increases RANTES/CCL5 expression through the NF-kB pathway in CD8þ T cells, CD4þ T cells, and monocytes/macrophages.29 A recent study showed that TNF-like weak inducer of apoptosis (TWEAK), a member of the TNF family, can act as a regulator of NF-kB activation, enhance RANTES/CCL5 and IL-8/CXCL8 production in HMEC-1 cells, and promote leukocyte migration. Moreover, serum levels of TWEAK were found to correlate with the severity of HSP.30 Although serum RANTES levels in the acute stage of HSP did not correlate with disease severity in our study (data not shown), the association between RANTES expression in tissues and disease severity needs further research. The major limitation of our study is the small sample size, which makes it underpowered to detect associations of the modest effects of gene polymorphisms. Our data show that the MCP1 2518 C/T gene polymorphism was a risk factor for susceptibility to HSP in ethnically Chinese children (power Z 0.7). Our results need to be validated in further large scale studies. In conclusion, chemokines are important in leukocyte recruitment to the inflammatory site, and they may promote vascular damage in vasculitis. Our results show a significant elevation of chemokines during the acute stage of HSP. MCP1/CCL2 2518T was associated with the development of HSP. RANTES/CCL5 gene polymorphisms may be associated with disease severity. RANTES/CCL5 403T carried an increased risk for HSP nephritis.
28 403 28 403
Genotype
Model 1a:ORs (95% CI)
p value
Model 2b: ORs (95% CI)
p
GGþCG CTþTT GGþCG CTþTT
1.85 2.97 0.98 2.7
0.38 0.038 0.968 0.037
1.03 2.58 0.59 4.33
0.97 0.11 0.41 0.009
(0.48e7.14) (1.06e8.33) (0.32e2.98) (1.06e6.85)
(0.23e4.63) (0.82e8.13) (0.17e2.08) (1.44e12.99)
CI Z confidence interval; HSP Z HenocheScho ¨nlein purpura; OR Z odds ratio. a Model 1: Crude ORs and 95% CIs. b Model 2: Adjusted for age, sex, RANTES/CCL5 28, RANTES/CCL5 403, and MCP1/CCL2 2518 genotype.
Please cite this article in press as: Yu H-H, et al., Chemokine MCP1/CCL2 and RANTES/CCL5 gene polymorphisms influence HenocheScho ¨nlein purpura susceptibility and severity, Journal of the Formosan Medical Association (2013), http://dx.doi.org/10.1016/ j.jfma.2012.12.007
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Financial support None.
Acknowledgments This study was supported by NRPGM, Microarray and SNP Core Facility for Genomic Medicine and Research Grants from the National Taiwan University Hospital (NTUH 096000698).
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H.-H. Yu et al. 13. Yang YH, Lai HJ, Kao CK, Lin YT, Chiang BL. The association between transforming growth factor-beta gene promoter C-509T polymorphism and Chinese children with HenocheSchonlein purpura. Pediatr Nephrol 2004;19:972e5. 14. Mantovani A, Bonecchi R, Locati M. Tuning inflammation and immunity by chemokine sequestration: decoys and more. Nat Rev Immunol 2006;6:907e18. 15. Wong M, Silverman ED, Fish EN. Evidence for RANTES, monocyte chemotactic protein-1, and macrophage inflammatory protein-1 beta expression in Kawasaki disease. J Rheumatol 1997;24:1179e85. 16. Bozoglu E, Dinc A, Erdem H, Pay S, Simsek I, Kocar IH. Vascular endothelial growth factor and monocyte chemoattractant protein-1 in Behcet’s patients with venous thrombosis. Clin Exp Rheumatol 2005;23:S42e8. 17. Ellingsen T, Elling P, Olson A, Elling H, Baandrup U, Matsushima K, et al. Monocyte chemoattractant protein 1 (MCP-1) in temporal arteritis and polymyalgia rheumatica. Ann Rheum Dis 2000;59:775e80. 18. Wu JF, Wu TC, Chen CH, Ni YH, Chen HL, Hsu HY, et al. Serum levels of interleukin-10 and interleukin-12 predict early, spontaneous hepatitis B virus e antigen seroconversion. Gastroenterology 2010;138. 165-72. e1-3. 19. Yang YH, Hung CF, Hsu CR, Wang LC, Chuang YH, Lin YT, et al. A nationwide survey on epidemiological characteristics of childhood HenocheSchonlein purpura in Taiwan. Rheumatology (Oxford) 2005;44:618e22. 20. Chen T, Guo ZP, Jiao XY, Jia RZ, Zhang YH, Li JY, et al. CCL5, CXCL16, and CX3CL1 are associated with HenocheSchonlein purpura. Arch Dermatol Res 2011;303:715e25. 21. Tahan F, Dursun I, Poyrazoglu H, Gurgoze M, Dusunsel R. The role of chemokines in Henoch Schonlein Purpura. Rheumatol Int 2007;27:955e60. 22. Chung HS, Kim HY, Kim HS, Lee HJ, Yuh JH, Lee ES, et al. Production of chemokines in Kawasaki disease, HenocheSchonlein purpura and acute febrile illness. J Korean Med Sci 2004;19: 800e4. 23. Yang YH, Lai HJ, Huang CM, Wang LC, Lin YT, Chiang BL. Sera from children with active HenocheSchonlein purpura can enhance the production of interleukin 8 by human umbilical venous endothelial cells. Ann Rheum Dis 2004;63:1511e3. 24. Kim HL, Lee DS, Yang SH, Lim CS, Chung JH, Kim S, et al. The polymorphism of monocyte chemoattractant protein-1 is associated with the renal disease of SLE. Am J Kidney Dis 2002; 40:1146e52. 25. Gonzalez E, Rovin BH, Sen L, Cooke G, Dhanda R, Mummidi S, et al. HIV-1 infection and AIDS dementia are influenced by a mutant MCP-1 allele linked to increased monocyte infiltration of tissues and MCP-1 levels. Proc Natl Acad Sci U S A 2002;99:13795e800. 26. Rovin BH, Lu L, Saxena R. A novel polymorphism in the MCP-1 gene regulatory region that influences MCP-1 expression. Biochem Biophys Res Commun 1999;259:344e8. 27. Mori H, Kaneko Y, Narita I, Goto S, Saito N, Kondo D, et al. Monocyte chemoattractant protein-1 A-2518G gene polymorphism and renal survival of Japanese patients with immunoglobulin A nephropathy. Clin Exp Nephrol 2005;9:297e303. 28. Nickel RG, Casolaro V, Wahn U, Beyer K, Barnes KC, Plunkett BS, et al. Atopic dermatitis is associated with a functional mutation in the promoter of the CeC chemokine RANTES. J Immunol 2000;164:1612e6. 29. Liu H, Chao D, Nakayama EE, Taguchi H, Goto M, Xin X, et al. Polymorphism in RANTES chemokine promoter affects HIV-1 disease progression. Proc Natl Acad Sci U S A 1999;96:4581e5. 30. Chen T, Guo ZP, Li MM, Li JY, Jiao XY, Zhang YH, et al. Tumour necrosis factor-like weak inducer of apoptosis (TWEAK), an important mediator of endothelial inflammation, is associated with the pathogenesis of HenocheSchonlein purpura. Clin Exp Immunol 2011;166:64e71.
Please cite this article in press as: Yu H-H, et al., Chemokine MCP1/CCL2 and RANTES/CCL5 gene polymorphisms influence HenocheScho ¨nlein purpura susceptibility and severity, Journal of the Formosan Medical Association (2013), http://dx.doi.org/10.1016/ j.jfma.2012.12.007
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Journal of the Formosan Medical Association (2013) xx, 1e6
Available online at www.sciencedirect.com
journal homepage: www.jfma-online.com
ORIGINAL ARTICLE
Chemokine MCP1/CCL2 and RANTES/CCL5 gene polymorphisms influence ¨nlein purpura susceptibility HenocheScho and severity Hsin-Hui Yu a, Pi-Hua Liu b, Yao-Hsu Yang a, Jyh-Hong Lee a, Li-Chieh Wang a, Wei-J. Chen c,d, Bor-Luen Chiang a,e,* a
Department of Pediatrics, National Taiwan University Hospital, Taipei, Taiwan, ROC Clinical Informatics and Medical Statistics Research Center, Chang Gung University, Tao-Yuan, Taiwan, ROC c Institute of Epidemiology, College of Public Health, National Taiwan University, Taipei, Taiwan, ROC d Genetic Epidemiology Core Laboratory, Research Center for Medical Excellence, National Taiwan University, Taipei, Taiwan, ROC e Department of Medical Research, National Taiwan University Hospital, Taipei, Taiwan, ROC b
Received 21 June 2012; received in revised form 26 November 2012; accepted 21 December 2012
KEYWORDS chemokine; HenocheScho ¨nlein Purpura (HSP); MCP1/CCL2; polymorphism; RANTES/CCL5
Background/Purpose: HenocheScho ¨nlein purpura (HSP) is the most common small vessel vasculitis in children. It is considered to be an IgA-containing immune complex-mediated disease. Chemokines are small secreted proteins that attract leukocytes during inflammation. Our aim was to determine the serum levels of chemokines and investigate the association of chemokine gene polymorphisms with childhood HSP. Materials and methods: Serum levels of chemokines (interleukin-8/CXCL8, MCP-1/CCL2, RANTES/CCL5, MIG/CXCL9, and IP-10/CXCL10) were determined using cytometric beads arrays. We investigated the association of three single-nucleotide polymorphisms (SNPs) MCP1/CCL2 2518C/T, RANTES/CCL5 403C/T, and RANTES/CCL5 28C/G with HSP in 85 HSP patients and 136 healthy controls. Results: Five serum chemokine levels were significantly elevated in patients with the acute stage of HSP compared to the normal controls (p < 0.05). MCP1/CCL2 2518 TT genotype and T allele were associated with the risk for HSP with OR (95% CI) 3.32 (1.45e7.59) and 1.78 (1.20e2.64), respectively. The RANTES/CCL5 28 GG genotype was associated with a significantly lower percentage of corticosteroid usage and lower corticosteroid accumulative
* Corresponding author. Department of Medical Research, National Taiwan University Hospital, Number 7, Chung-Shan South Road, Zhongzheng District, Taipei City 100, Taiwan, ROC. E-mail address: [email protected] (B.-L. Chiang). 0929-6646/$ - see front matter Copyright ª 2013, Elsevier Taiwan LLC & Formosan Medical Association. All rights reserved. http://dx.doi.org/10.1016/j.jfma.2012.12.007
Please cite this article in press as: Yu H-H, et al., Chemokine MCP1/CCL2 and RANTES/CCL5 gene polymorphisms influence HenocheScho ¨nlein purpura susceptibility and severity, Journal of the Formosan Medical Association (2013), http://dx.doi.org/10.1016/ j.jfma.2012.12.007
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H.-H. Yu et al. dose in HSP patients. RANTES/CCL5 403 TC and TT genotype were significantly associated with renal manifestations with an OR (95% CI) of 4.33 (1.44e12.99), adjusted for sex and age and the other two SNP genotypes. Conclusion: Our results support the fact that chemokines play important roles in the pathogenesis of HSP. MCP1/CCL2 gene polymorphisms were associated with susceptibility for HSP. RANTES/CCL5 gene polymorphisms may be related to disease severity and HSP nephritis. Copyright ª 2013, Elsevier Taiwan LLC & Formosan Medical Association. All rights reserved.
Introduction HenocheScho ¨nlein purpura (HSP) is the most common small vessel vasculitis that occurs in children. It is an IgAcontaining immune complex-mediated vasculitis, which is triggered by common viral or bacterial infections.1 The European League Against Rheumatism (EULAR) proposed consensus criteria for the classification of HSP in 2010, which require individuals to have purpura or petechiae with lower limb predominance and at least one of the four following criteria: abdominal pain, histopathology (typical leukocytoclastic vasculitis or proliferative glomerulonephritis with predominant IgA deposits), arthritis or arthralgia, or renal involvement (proteinuria > 0.3 g/day or hematuria or red blood cell casts >5 red blood cells/high power field, or 2þ on a dipstick).2 Perivascular accumulation of neutrophils in the acute inflammatory lesions is the typical histopathological feature of HSP, known as leukocytoclastic vasculitis. Lymphocyte and monocyte/macrophage infiltration and neovascularization are found later in the disease course.3 The mechanisms underlying the perivascular leukocyte infiltrates and endothelial damage in HSP are not fully understood. Previous studies have shown the elevation of inflammatory mediators in the serum of HSP patients, including tumor necrosis factor (TNF)-a, interleukin-6, transforming growth factor (TGF)-b, and vascular endothelial growth factor (VEGF).47 Genetic variations in key inflammatory genes, including interleukin-1 beta (IL1B), interleukin-1 receptor antagonist (IL1RA), intercellular adhesion molecule-1 (ICAM1), IL8, VEGF, and TGFB gene polymorphisms also contribute to the susceptibility or different clinical manifestations in HSP.813 Proinflammatory chemokines are a group of small secreted proteins that attract leukocytes to the inflammatory site and then activate them. It is a critical pathogenic factor in tissue damage during the development of vasculitis. Chemokine monocyte chemoattractant protein 1 (MCP-1/CCL2), and interferon gamma-induced protein-10 (IP-10/CXCL10) are strong chemoattractants for monocytes/macrophages. Regulated upon activation, normal Tcells expressed and secreted RANTES/CCL5, monokine induced by gamma interferon (MIG/CXCL9), and IP-10/ CXCL10 are chemoattractants for T cells.14 The roles of chemokines have been investigated in many rheumatic diseases and vasculitis, such as Kawasaki disease, Behcet’s disease, and polymyalgia rheumatica.1517 However, the roles of proinflammatory chemokines except IL-8 have not been investigated in HSP before. We therefore aimed to investigate the changes in serum levels of five different proinflammatory chemokines during
the acute and convalescent stages of HSP. We tested the association between serum chemokines levels at the acute stage of HSP and disease severity. We also tested the hypothesis that single nucleotide polymorphisms (SNPs) in the MCP1/CCL2 and RANTES/CCL5 genes would be associated with serum chemokine levels, disease susceptibility, or severity.
Materials and methods Study subjects This study was conducted at National Taiwan University Hospital, a tertiary referral center. Eighty-five patients who were diagnosed with HSP, and 136 volunteersdhealthy, sex-matched children (73 boys and 63 girls) aged 2e18 yearsdwere enrolled. The diagnosis of HSP was based on the criteria defined by EULAR in 2010. Disease severity was clinically assessed by the degree of systemic involvement. Gastrointestinal involvement was defined as abdominal pain with/without vomiting, and/or stool occult blood 1þ, and/or melena. Renal involvement was defined as per the EULAR criteria of 2010. Informed consent and institutional approval were obtained.
Serum and cytometric bead array Serum was collected from 63 of the 136 age-matched healthy controls, and 55 of 85 HSP patients in the acute stage of HSP. Twenty HSP patients also donated blood during the convalescent stage of HSP. The convalescent stage was defined as the stage in which there was complete resolution of symptoms and signs for 1 month after discontinuing medication. Serum concentrations of MIG/ CXCL9, IP-10/CXCL10, MCP-1/CCL2, RANTES/CCL5 and IL8/CXCL8 were analyzed by Human Chemokine Kit I Cytometric Beads Array kits from BD Biosciences (San Diego, CA, USA; BD CBA), according to the manufacturer’s recommended protocols. The serum samples for RANTES/CCL5 were diluted 1:100 using the assay diluent to fit the concentration range.
DNA extraction and single nucleotide polymorphism (SNP) genotyping Genomic DNA of the 85 HSP patients and 136 controls was extracted from the anticoagulated blood using a commercially available DNA extraction kit (QIAGEN, QIAamp DNA Blood Mini Kit, Germany) and stored at 20 C
Please cite this article in press as: Yu H-H, et al., Chemokine MCP1/CCL2 and RANTES/CCL5 gene polymorphisms influence HenocheScho ¨nlein purpura susceptibility and severity, Journal of the Formosan Medical Association (2013), http://dx.doi.org/10.1016/ j.jfma.2012.12.007
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Chemokine MCP1 and RANTES gene polymorphisms in HSP before testing. We used the candidate gene approach by selecting well-investigated SNPs in chemokine genes (MCP1/CCL2 2518 C/T (dbSNP ID rs1024611), RANTES/ CCL5 403 C/T (dbSNP ID rs2107538), and RANTES/ CCL5 28 C/G (dbSNP ID rs2280788) in a case control study design. We chose these three previously identified common functional variants from the National Center for Biotechnology Information (NCBI) database (http://www.ncbi. nlm.nih.gov/gene). Genotypes were identified by multiplex polymerase chain reaction (PCR) and SNP analyses with the GenomeLab SNPstream genotyping platform (Beckman Coulter Inc. Fullerton, CA) and its accompanying SNPstream software suite. The primers for the multiplex PCR and single-base extension primers (tagged SNP primers) were designed by the software provided by Beckman Coulter Inc. The detailed methods, reagents, and PCR conditions were described in a previous report.18 The direct sequencing reactions using an ABI 3730 automated DNA sequencer (Applied Biosystems, Foster City, CA) in 10 cases and 10 controls resulted in identical genotypes by multiplex PCR and SNP analyses. The average genotype call rates by multiplex PCR were 95.29% and 98.92%, respectively, for the cases and controls. The overall genotype call rate was 98.32%.
Statistical methods The ManneWhitney U test was used to compare the serum chemokine levels between the groups of patients. A paired t test was used to compare serum chemokine levels in the acute and convalescent stage of HSP. A c2 goodnessof-fit test was performed using the SAS/Genetics module, version 9.1 (SAS Institute, Cary, NC) to assess whether the observed genotype frequencies of each SNP marker departed from the HardyeWeinberg equilibrium (HWE) in the HSP patients and control subjects separately. After the frequencies of the alleles and genotypes had been calculated, Armitage’s trend and genotypic case-control tests were determined with c2 tests using the SAS/Genetics module to assess each SNP for a global association with HSP. When the global association test was significant, an unconditional logistic regression was undertaken using SAS Proc LOGISTIC to calculate allele or genotype odds ratios (OR) with 95% confidence intervals (CIs) for the potential association between HSP and SNPs. Logistic regression models were used to test the association of genotypes with clinical outcomes. A p value of less than 0.05 was considered to be statistically significant. Power calculations were performed at a type 1 error rate (a value) of 0.05 and a prevalence of 0.0001 for HSP using the Genetic Power Calculator (http://pngu.mgh.harvard.edu/ wpurcell/gpc/). The prevalence was estimated from the annual incidence of 12.9 per 100,000 children in Taiwan.19
Results Patient characteristics The demographic data of the 85 children with HSP are summarized in Table 1. All 85 (100%) patients had purpura,
3 Table 1 Clinical characteristics and basic demographic data of the 85 HSP patients. HSP patients Sex (male:female) Age (years) Previous infection history Purpura Gastrointestinal involvement Arthritis or arthralgia Renal involvement Central nervous system involvement Recurrence Steroid treatment Disease duration of 6 months or more IgA elevation
46:39 (1.18:1) 6.4 2.7 51/84 (60.7%) 85/85 (100%) 62/85 (72.9%) 60/85 (70.6%) 30/85 (35.3%) 2/85 (2.4%) 9/85 (10.6%) 77/84 (91.7%) 14/85 (16.5%) 42/70 (60%)
of whom 29 (34.1%) had prolonged purpura for 4 weeks or more. Sixty-two (72.9%) patients had gastrointestinal (GI) involvement, of whom 18 (21.2%) had GI bleeding. Thirty (35.3%) patients had renal involvement, including 14 (16.5%) with severe nephropathy (nephrotic syndrome or impaired renal function) and three (3.5%) with renal sequelae (persistent urine abnormalities over 2 years). Sixty percent of the HSP patients had serum IgA elevation at disease onset.
Chemokine expressions in the HSP patients and controls The serum chemokine levels of MIG/CXCL9, IP-10/CXCL10, MCP-1/CCL2, RANTES/CCL5 and IL-8/CXCL8 were significantly elevated in patients with the acute stage of HSP compared to the normal controls (p < 0.05) (Fig. 1). Compared with the acute stage, serum chemokine levels decreased significantly in the convalescent stage of HSP in 20 patients (p < 0.05), except for IL-8/CXCL8 (p Z 0.109) and MIG/CXCL9 (p Z 0.805) (Fig. 2). There was more renal involvement in the HSP patients with persistently elevated
Figure 1 Chemokine serum concentrations in the controls and HSP patients in the acute stage. The serum chemokine levels of MIG/CXCL9, IP-10/CXCL10, MCP-1/CCL2, RANTES/ CCL5, and IL-8/CXCL8 are significantly elevated in the acute stage of HSP compared to the normal controls (p < 0.05). HSP Z HenocheScho ¨nlein purpura (*p < 0.01, **p < 0.0001).
Please cite this article in press as: Yu H-H, et al., Chemokine MCP1/CCL2 and RANTES/CCL5 gene polymorphisms influence HenocheScho ¨nlein purpura susceptibility and severity, Journal of the Formosan Medical Association (2013), http://dx.doi.org/10.1016/ j.jfma.2012.12.007
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4 MIG/CXCL9 levels in the convalescent stage than that of the HSP patients with decreased MIG/CXCL9 levels in the convalescent stage (71.4% vs. 16.7%) (p Z 0.045).
Polymorphisms and HSP risk Three SNPs and RANTES/CCL5 and MCP1/CCL2 genes were investigated. None of the SNP distributions showed a deviation from the HardyeWeinberg equilibrium. Table 2 presents the genotypes of the three SNPs for HSP patients (n Z 85) and controls (n Z 136), as well as ORs for each of the SNPs. MCP1 2518 TT and the TC genotype carried an increased risk for HSP (OR (95% CI) 3.32 (1.45e7.59) and 2.0 (1.03e3.9), respectively). After adjusting for age and sex, the adjusted OR (95% CI) of MCP1 2518 TT and TC genotype remained unchanged: 3.32 (1.45e7.59) and 2.01 (1.03e3.92), respectively. The MCP1 2518 T allele carried an increased risk for HSP with an allelic OR (95% CI) of 1.78 (1.20e2.64), p < 0.0001.
Chemokine expressions in the HSP patients with different genotypes Serum RANTES/CCL5 and MCP-1/CCL2 levels in the acute stage of HSP did not significantly correlate with the corresponding genotype (data not shown).
Factors associated with clinical outcomes in the HSP patients Patients with the RANTES/CCL5 28 G/G and C/G genotype had a significantly lower percentage of corticosteroid usage and lower corticosteroid accumulative dose (median dose 11.3 mg/kg vs. 20.5 mg/kg) (p Z 0.04) than patients carrying the RANTES/CCL5 28C allele. The RANTES/ CCL5 403 TC and TT genotypes were associated with GI
H.-H. Yu et al. and renal involvement in the HSP patients (OR (95% CI) 2.97 (1.06e8.33) and 2.7 (1.06e6.85), respectively). However, using multivariate logistic regression models adjusting for age, sex, RANTES/CCL5 28G, RANTES/CCL5 403T, and MCP1/CCL2 2518T genotypes, only the RANTES/ CCL5 403 TC and TT genotypes were associated with renal involvement (OR (95% CI) 4.33 (1.44e12.99), p Z 0.009). There was no significant association between clinical outcomes and the RANTES/CCL5 28 GG and CG genotypes (Table 3).
Discussion Our results showed significant elevations of the chemokines MIG/CXCL9, IP-10/CXCL10, MCP-1/CCL2, RANTES/CCL5, and IL-8/CXCL8 in the sera of patients with the acute stage of HSP. Our finding suggests that proinflammatory chemokines, which function in the recruitment of different types of leukocytes, are involved in the pathogenesis of HSP. Our results are consistent with a recent study that showed elevation of serum levels of RANTES/CCL5, CXCL16, CX3CL1 in the acute stage of HSP,20 but contradictory to previous reports of decreased IP-10/CXCL10 serum level or no elevation of IP-10/CXCL10, MCP-1/CCL2, and Gro-a/CXCL1 levels in acute HSP.21,22 Recent studies have shown that chemokines are produced during inflammatory stimuli from endothelial cells. Yang et al found that IgA antiendothelial cell antibodies in the serum of HSP patients could stimulate human umbilical vein endothelial cells (HUVECs) through extracellular signalregulated kinase (ERK) pathways to produce IL-8/CXCL8.6,23 Chen et al further showed that sera from patients with active HSP could induce MCP-1/CCL2, RANTES/CCL5, IL-8/ CXCL8, CXCL16, and CX3CL1 production in HUVECs or the human dermal microvascular endothelial cell line (HMEC-1)
Figure 2 Chemokine serum concentrations in the HSP patients in the acute and convalescent stage. The serum MCP-1, RANTES, and IP-10 levels decrease significantly in the convalescent stage (p < 0.05). HSP Z HenocheScho ¨nlein purpura. Please cite this article in press as: Yu H-H, et al., Chemokine MCP1/CCL2 and RANTES/CCL5 gene polymorphisms influence HenocheScho ¨nlein purpura susceptibility and severity, Journal of the Formosan Medical Association (2013), http://dx.doi.org/10.1016/ j.jfma.2012.12.007
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Chemokine MCP1 and RANTES gene polymorphisms in HSP Table 2
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Association analysis between the SNPs of the MCP1/CCL2 and RANTES/CCL5 genes and HSP.
Gene
SNP genotype
MCP1
2518 C/T
RANTES
403 C/T
RANTES
28 C/G
TT TC CC TT TC CC GG GC CC
HSPa (n Z 85)
Controla (n Z 136)
ORsb
95% CIb
pb
21 44 17 10 36 39 2 15 68
19 66 51 13 54 69 7 22 107
3.32 2.0 1 0.92 1.09 1 0.33 1.15 1
1.45e7.59 1.03e3.90 d 0.34e2.53 0.56e2.12 d 0.03e3.29 0.51e2.62 d
0.005 0.04 d 0.88 0.80 d 0.35 0.73 d
(25.6%) (53.7%) (20.7%) (11.8%) (42.4%) (45.9%) (2.4%) (17.6%) (80%)
(14.0%) (48.5%) (37.5%) (9.6%) (39.7%) (50.7%) (5.1%) (16.2%) (78.7%)
CI Z confidence interval; HSP Z HenocheScho ¨nlein purpura; OR Z odds ratio; SNP: single nucleotide polymorphism. a The number of the patients along with the percentage in brackets. b Logistic regression models are used to calculate OR and 95%CIs, and the corresponding p values for each genotype.
through ERK and nuclear factor-kB (NF-kB) pathways. Patients’ sera-stimulated HMEC-1 supernatants were also noted to enhance leukocytes migration.20 These results support the important role of chemokines in perivascular leukocyte infiltrates and the vascular damage in HSP. The genetic basis of HSP is unclear. To the best of our knowledge, this is the first study to report an association of MCP1/CCL2 2518T and the risk of susceptibility to HSP. MCP-1 expression is inducible by proinflammatory cytokines (TNF-a, IL-1b) through NF-kB in various cell types. The gene expression of the MCP1/CCL2 2518C/T polymorphism in the promoter region is controversial. Kim et al reported that the T allele at 2518 was associated with upregulation of MCP-1/CCL2.24 In contrast, Rovin et al and Gonzalez et al reported that MCP1/CCL2 2518C produced more MCP1.25,26 These discrepancies may be related to differences in disease or ethnicity. The MCP1/CCL2 2518TT genotype has been shown to be an independent risk factor for the progression of renal diseases in Japanese patients with IgA nephropathy, which shares a common disease mechanism with HSP nephritis.27 To elucidate the role of the MCP1/ CCL2 2518 gene polymorphism in HSP, the relationship between the MCP1/CCL2 2518 genotype, MCP-1 production from endothelial cells, and macrophage infiltration in skin and other target organs such as the kidney should be investigated in future studies. Our results showed that the RANTES/CCL5 403 TC and TT genotypes carried an increased risk of HSP nephritis. RANTES/CCL5 403 C to T change results in a new binding element for GATA transcriptional factors and has higher transcriptional activity.28 The promoter of that RANTES/
Table 3
Frequency and ORs of RANTES/CCL5 genotypes in HSP patients with different disease manifestations.
HSP involvement
SNP
GI
RANTES RANTES RANTES RANTES
Renal
CCL5 28 gene polymorphism site is close to the NF-kB binding site, which is located at position 32 and plays a critical role in the upregulation of RANTES promoter activity. Liu et al reported that RANTES/CCL5 28G increases RANTES/CCL5 expression through the NF-kB pathway in CD8þ T cells, CD4þ T cells, and monocytes/macrophages.29 A recent study showed that TNF-like weak inducer of apoptosis (TWEAK), a member of the TNF family, can act as a regulator of NF-kB activation, enhance RANTES/CCL5 and IL-8/CXCL8 production in HMEC-1 cells, and promote leukocyte migration. Moreover, serum levels of TWEAK were found to correlate with the severity of HSP.30 Although serum RANTES levels in the acute stage of HSP did not correlate with disease severity in our study (data not shown), the association between RANTES expression in tissues and disease severity needs further research. The major limitation of our study is the small sample size, which makes it underpowered to detect associations of the modest effects of gene polymorphisms. Our data show that the MCP1 2518 C/T gene polymorphism was a risk factor for susceptibility to HSP in ethnically Chinese children (power Z 0.7). Our results need to be validated in further large scale studies. In conclusion, chemokines are important in leukocyte recruitment to the inflammatory site, and they may promote vascular damage in vasculitis. Our results show a significant elevation of chemokines during the acute stage of HSP. MCP1/CCL2 2518T was associated with the development of HSP. RANTES/CCL5 gene polymorphisms may be associated with disease severity. RANTES/CCL5 403T carried an increased risk for HSP nephritis.
28 403 28 403
Genotype
Model 1a:ORs (95% CI)
p value
Model 2b: ORs (95% CI)
p
GGþCG CTþTT GGþCG CTþTT
1.85 2.97 0.98 2.7
0.38 0.038 0.968 0.037
1.03 2.58 0.59 4.33
0.97 0.11 0.41 0.009
(0.48e7.14) (1.06e8.33) (0.32e2.98) (1.06e6.85)
(0.23e4.63) (0.82e8.13) (0.17e2.08) (1.44e12.99)
CI Z confidence interval; HSP Z HenocheScho ¨nlein purpura; OR Z odds ratio. a Model 1: Crude ORs and 95% CIs. b Model 2: Adjusted for age, sex, RANTES/CCL5 28, RANTES/CCL5 403, and MCP1/CCL2 2518 genotype.
Please cite this article in press as: Yu H-H, et al., Chemokine MCP1/CCL2 and RANTES/CCL5 gene polymorphisms influence HenocheScho ¨nlein purpura susceptibility and severity, Journal of the Formosan Medical Association (2013), http://dx.doi.org/10.1016/ j.jfma.2012.12.007
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Financial support None.
Acknowledgments This study was supported by NRPGM, Microarray and SNP Core Facility for Genomic Medicine and Research Grants from the National Taiwan University Hospital (NTUH 096000698).
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Please cite this article in press as: Yu H-H, et al., Chemokine MCP1/CCL2 and RANTES/CCL5 gene polymorphisms influence HenocheScho ¨nlein purpura susceptibility and severity, Journal of the Formosan Medical Association (2013), http://dx.doi.org/10.1016/ j.jfma.2012.12.007
